Human arginase and site-directed pegylated human arginase and the use thereof

ABSTRACT

The present invention provides a site-directed mutated arginase and the preparation method thereof, and the use of said site-directed mutated arginase in preparing a medicament for treating an arginase-related disease. The present invention also provides a site-directed pegylated arginase and the preparation method thereof, and the use of said pegylated arginase in preparing a medicament for treating an arginase-related disease.

This application claims priorities from the Chinese Patent Application No. 201110445965.8, filed on Dec. 27, 2011, entitled “Human arginase and pegylated human arginase and the use thereof”, and from the Chinese Patent Application No. 201210069605.7, filed on Mar. 16, 2012, entitled “Human arginase and site-directed pegylated human arginase and the use thereof”, which are incorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

The hard copy of the sequence listing submitted herewith and the corresponding computer readable form are both incorporated herein by reference in their entireties.

FIELD OF INVENTION

The present invention provides an arginase, the preparation method thereof and the use thereof in the treatment of arginase-related diseases. Specifically, the present invention provides a site-directed mutated arginase, the preparation method thereof and the use thereof in the treatment of arginase-related diseases. The present invention also provides a site-directed pegylated arginase, the preparation method thereof and the use thereof.

BACKGROUND OF THE INVENTION

Arginine is an important amino acid in mammals including human, which is involved in various physiological processes, including cell proliferation and growth. For example, arginine is an immediate precursor for the synthesis of the potential signal molecule nitric oxide (NO). NO functions as a neurotransmitter, a muscle relaxant or a vasodilator. The biosynthesis of NO involves nitric oxide synthase catalyzed Ca⁺⁺ and NADPH-dependent reactions. Another function of arginine is as a precursor for polyamine, spermidine or spermine and participates in different physiological processes.

Studies showed that when arginine is less than 8 μM, cancer cells undergo irreversible death (Srorr & Burton, 1974, The effects of arginine deficiency on lymphoma cells. Br. J. Cancer 30, 50). By studying the effect on cell growth, it was found that upon removal of arginine, the normal cells in the cell cycle G0 phase would enter a resting state and remain viable for several weeks without significant damage; when the concentration of arginine returned to normal level, the cells would return to normal cell cycle. However certain tumor cells would proceed from the ‘R’ point of the cell cycle G1 phase to S phase at deprivation of arginine, and undergo apoptosis soon. The apoptosis of tumor cells as a result of arginine deficiency is irreversible. Therefore, scientists began to consider treating cancers by controlling the level of arginine in the body, especially for auxotrophic tumors such as liver cancer and melanoma. This approach has now been used to study the inhibition of various tumors.

Arginase is the enzyme catalyzing the hydrolysis of L-arginine to ornithine and urea. In general, arginase is expressed in liver, kidney and testis of the urea-producing animals (mammals, elasmobranchs, amphibians, and turtles) as one of the enzymes in the urea cycle. Arginase catalyzes the final step of the urea recycle pathway in mammals, converting arginine to ornithine and urea. In most mammals, the family of arginase includes arginase I and arginase II. Arginase I is mainly expressed in the liver cells, and arginase II is mainly expressed in the kidney and erythrocytes.

There are two methods to produce arginase; of which one is to separate it from the arginase producing organism, and the other is through recombinant genetic engineering techniques. The latter has its advantages. For example, experiments have showed that a great amount of arginase could be produced by E. coli. However, there may be technical problems to produce arginase through recombinant genetic engineering techniques, such as low enzyme activity or poor stability and short half-life in vivo, limiting its application in clinical practice.

U.S. Pat No. 7,951,366B2 disclosed a pharmaceutical composition and method for the treatment of human malignant tumor by arginine depletion using recombinant human arginase I with his-tag, wherein the arginase is modified by covalently conjugating with polyethylene glycol of molecular weight of 5,000 (MW 5,000) at the N-terminal or the amine group on the surface of the arginase. The modified human arginase had an increased stability with a half-life of 3 days in human serum.

US20100247508A1 disclosed a modified human arginase with his-tag, wherein the 168 and 303 cysteines are replaced with serines and the arginase is pegylated with polyethylene glycol of a molecular weight of 20 KDa. Like the recombinant human arginase I disclosed in U.S. Pat. No. 7,951,366B2, an additional peptide fragment, His-tag is included in the amino acid sequence of the arginase. The drug regulatory agencies in most countries do not recommend the use of these peptide fragments. For example, China State Food and Drug Administration indicates in the “Technical Quidelines on the Quality Control of Recombinant DNA Product for Human Use” that additional peptide fragments such as His-tag introduced for the purpose of simplifying the production process should be removed as far as possible from the final product.

WO 2011/008495A2 disclosed a site-directed modified arginase, in which the three cysteines were retained and another cysteine residue is introduced substituting the third amino acid residue of the N-terminus, and then pegylated with methoxy polyethylene glycol maleimide of molecular weight of 20 KDa. Since the said human arginase still carries the original three cysteine residues, its pegylation product is prone to heterogeneous and low in yield, which also adds difficulties for purification.

The present technical field has the need for a better arginase or derivatives thereof, for the treatment of arginine related diseases or disorders.

SUMMARY OF THE INVENTION

The present invention provides an isolated and substantially pure arginase. Arginase is the enzyme in the final step of the urea recycle pathway in the synthesis of urea in mammals, converting arginine to ornithine and urea. In most mammals, the family of arginase includes arginase I and arginase II. Studies on arginase I of various origins showed that arginase I of different origins have a lot of conserved regions and active sites, though their sequence may be different. As reported by Haraguchi, Y., etc., 1987, Proc. Natl. Acad. Sci. 84, 412-415, wild-type human arginase I has an amino acid sequence of SEQ ID NO.1, which comprises three cysteines at amino acid position 45, 168 and 303. The wild-type human arginase I has a nucleic acid sequence of SEQ ID NO. 2.

In the present invention, the term “isolated” refers to a non-natural form. The term “substantial pure” means that the product may comprise other components derived from production or protein modification, yet such other components are substantially not present or only present in a small ratio.

In the present invention, the description on the location of amino acid site is commonly used by the skilled in the art. For example, the phrase “in position 45 of the sequence of SEQ ID NO.x” or “Cys (cysteine) in position 45 of the sequence of SEQ ID NO.x” or “Cys45” all refers to the 45^(th) amino acid residue in the amino sequence or the cysteine in position 45.

In one aspect, the present invention provides a mutant arginase, which is a human arginase I comprising

-   -   (1) an amino acid sequence of SEQ ID NO:1, wherein any one, two         or three of the cysteines at positions 45, 168 and 303 is/are         mutated, or     -   (2) an amino acid sequence wherein substitution, deletion,         insertion, addition or inversion of one or more amino acids is         introduced in the amino acid sequence defined in (1),

and which has arginase activity.

In one aspect, the cysteines in the arginase of the present invention are mutated to non-polar amino acids. According to the properties of their side chain, the amino acids can be classified as polar or non-polar amino acids. Amino acids having side chains being uncharged or weakly polarized are called non-polar amino acids, such as, glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline.

In one aspect, the cysteines in the arginase of the present invention are independently mutated to glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan or proline. Preferably, the cysteines are independently mutated to alanine. The present invention has unexpectedly found that the enzymatic activity of human arginase I is significantly increased when the cysteine, which is a non-ionic polar amino acid is mutated to a non-polar amino acid (such as alanine). One of the possible reasons is the binding between the mutant arginase and the substrate is enhanced.

In one aspect, one of the cysteines at positions 45, 168 and 303 of the arginase of the present invention is mutated. In a further aspect of the present invention, cysteine at position 303 is mutated. Preferably, said cysteine is mutated to glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan or proline; and more preferably, said cysteine is mutated to alanine.

In one aspect, any two of the cysteines at positions 45, 168 and 303 of the arginase of the present invention are mutated. In a further aspect of the present invention, cysteines at positions 45 and 303 are mutated. Preferably, said cysteines are mutated to glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan or proline; and more preferably, said cysteins are mutated to alanine. In one aspect, cysteines at positions 168 and 303 of the amino acid sequence of SEQ ID NO. 1 in the arginase of the present invention are mutated. Preferably, said cysteines are mutated to glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan or proline; and more preferably, said cysteines are mutated to alanine. In one aspect, cysteines at positions 45 and 303 of the amino acid sequence of SEQ ID NO. 1 in the arginase of the present invention are mutated. Preferably, said cysteines are mutated to glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan or proline; and more preferably, said cysteines are mutated to alanine.

In one aspect, cysteines at positions 45, 168 and 303 of the amino acid sequence of SEQ ID NO. 1 in the arginase of the present invention are mutated. Preferably, said cysteines are mutated to glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan or proline; and more preferably, said cysteines are mutated to alanine.

The arginase of the present invention has higher enzymatic activity than the wild-type arginase. The arginase of present invention generally has a specific activity of at least about 500 U/mg; preferably, the activity is at least about 700 U/mg; and more preferably, the activity is at least about 800 U/mg. Arginase of the present invention generally has higher enzymatic activity than the wild-type arginase.

In one aspect of the present invention, the arginase has a nucleotide sequence of SEQ ID NO. 2, wherein at least one codon for amino acid residue cysteine at position 45, 168 and 303 of the amino acid sequence of SEQ ID NO.1 is substituted. Preferably, one, two or three of the codons for cysteine is/are substituted. Amino acids are encoded by polynucleotides. A three-nucleotide codon in a messenger RNA defines a single amino acid. In one aspect of the present invention, the nucleotide substitution is the codon TGT substituted by a codon encoding for glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan or proline. Preferably, the codon TGT is substituted by a codon encoding for alanine.

In one aspect of the present invention, said codons encoding for alanine is GCT, GCC, GCA, GCG, and preferably is GCT.

In one aspect, the present invention provides a method for preparing the arginase mentioned above, including expressing the gene of the arginase or mutant arginase in a host which is able to express the protein. The preparation method for the arginase of present invention generally includes the following major steps. Desired genes or nucleotide fragments are obtained by PCR or synthetic method. The DNA fragment with the desired gene is linked with a vector, such as a plasmid, phage or virus, which can replicate independently and has a selection marker to form recombinant DNA molecules. The ligation of DNA fragments into a vector is mainly through homopolymer tails, cohesive ends, blunt-end or artificial linker. Recombinant DNA has to be introduced into the host cell in order to be multiplied and expressed. According to the different properties of the vectors, transfection, transformation, transduction are carried out so that the recombinant DNA molecules are introduced into a host cell and multiplied. Suitable genetic engineering hosts are well known in the art, which include Escherichia coli, yeast, insect cells and the like.

The present invention also provides an isolated and substantially pure pegylated arginase, characterized in that the pegylation is site-specific. In said pegylated arginase, polyethylene glycol molecule is covalently linked with the specified amino acid residues of the arginase to achieve the site-specific pegylation. In the present invention, each of the arginase is linked with at least one polyethylene glycol molecule to form the pegylated arginase.

In one aspect of the present invention, the arginase in said pegylated arginase is the mutant arginase as previously defined.

In one aspect, the present invention provides a pegylated arginase, wherein the arginase is a human arginase I which comprises

-   -   (1) an amino acid sequence of SEQ ID NO:1, wherein any one or         two of cysteines at positions 45, 168 and 303 is/are mutated, or     -   (2) an amino acid sequence wherein substitution, deletion,         insertion, addition or inversion of one or more amino acids is         introduced in the amino acid sequence defined in (1),

and which has arginase activity.

In one aspect, the cysteines in the pegylated arginase of the present invention are independently mutated to non-polar amino acid. The structures of the 20 amino acids are different due to the difference of their side chain. Amino acids having side groups being uncharged or weakly polarized belong to non-polar amino acids, such as, glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline.

In one aspect of the invention, the pegylated arginase I does not comprise a tag sequence for use in the purification, such as a His-tag sequence added at the C or N terminus. His-tag fusion protein is the most common means of protein expression, which has the advantage of easy for purification and without significant effect to the activity of the protein. Protein expression products either in soluble form or in inclusion body could be purified by immobilized metal ion affinity chromatography. On the other hand, because of the potential immunogenicity property, China State Food and Drug Administration indicates in the “Technical Quidelines on the Quality Control of Recombinant DNA Product for Human Use” that additional peptide fragments such as His-tag introduced for the purpose of simplifying the production process should be removed as far as possible from the final product.

In the present invention, the pegylation of the arginase can be achieved by chemical modification, said modification can be carried out by using a polyethylene glycol derivative with a coupling agent (also referred to as a pegylation reagent) to covalently link a polyethylene glycol molecule with a group on the arginase. Said chemical modification can be specific, i.e. conjugate PEG molecules with a specific group on the arginase by using coupling reagents with specific binding activities. Site-directed pegylation conforms to the following principles: (1) modification of the protein active site should be avoided as far as possible to prevent its negative effect on protein activity; and (2) the entire process should be as simple as possible to be controllable. Common polyethylene glycol molecule has a hydroxyl group at each end thereof. A methoxy polyethylene glycol (mPEG) is a polyethylene glycol blocked in one end with a methoxy group. The most commonly studied pegylation reagents used in the modification of peptides or proteins are derivatives of methoxy polyethylene glycol (mPEG). One of the common pegylation method is to covalently link polyethylene glycols or pegylation reagents with ε-NH₂ of surface lysine or N-terminal α-NH₂ of the protein, such as methoxy polyethylene glycol succinimidyl butyrate (mPEG-SBA), mPEG-succinimidyl propionate (mPEG-SPA), mPEG-succinimidyl succinate (mPEG-SS), mPEG-succinimidyl carbonate (mPEG-SC), mPEG-Succinimidyl Glutarate (mPEG-SG), mPEG-N-hydroxyl-succinimide (mPEG-NHS), mPEG-tresylate and mPEG-aldehyde. Another common pegylation method is to selectively use of polyethylene glycols or pegylation reagents which can specifically conjugated with the thiol group of proteins, such as mPEG-maleimide, mPEG-ortho-pyridyl-disulphide, mPEG-vinylsulfone and mPEG-iodoacetamide to modify the thiol group of peptides or proteins. Since the number of thiol groups in proteins or peptides is relative small, it is generally easy to achieve a homogeneous pegylation product.

Human arginase has three cysteine residues located at positions 45, 168 and 303 of the amino acid sequence. In one aspect of the present invention, said pegylated arginase has site-specific pegylation at one or two said cysteine positions.

In one aspect of the present invention, said site-specific pegylation is achieved by site-directed mutagenesis of the cysteine of the arginase and followed by site-specific pegylation of mutant arginase at cysteine residue. In a further aspect of the present invention, said cysteine-specific pegylation is achieved by covalently linking PEG with the thiol group of cysteine on the arginase by methoxy polyethylene glycol -maleimide.

In one aspect of the present invention, said site-specific pegylation is achieved by site-directed mutagenesis of cysteine to other amino acid at one or two cysteine positions which is/are not to be pegylated, and preferably mutated to alanine, glycine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan or proline; and more preferably, said cysteine(s) is/are mutated to alanine.

The selection of molecular weight of the PEG should make a comprehensive consideration of the bio-activity and the pharmacokinetics properties. Studies showed that the acting time of the modified protein drugs in the body is correlated with the numbers and molecule weight of PEG conjugated. The PEG used for the present invention has a molecule weight ranging from 5K to 40K, linear or branched. The PEG used for the present invention can be PEG derivatives used in the technical field. The PEG used for the present invention is not limited to certain specified types. In one aspect of the present invention, the average molecular weight of the PEG molecule covalently linked with the arginase is about 20K, 30K, or 40K. Pegylated proteins can be administrated by intravenous injection. The average molecular weight of the polyethylene glycol may affect the serum half-life thereof. Studies found that renal clearance of PEG depends on its glomerular filtration rate. The glomeruli can allow proteins with molecular weight smaller than 70 KDa or PEGs with molecular weight smaller than 30 KDa to be filtered. The Effect of molecular weight of PEG on the half-life and activity of the bound protein is usually unpredictable, which is in general closely related with the molecular weight of protein, mode of action, active site, and PEG-binding site of protein.

In one aspect of the present invention, the pegylated arginase is site-directed pegylated at positions 45 and 168 of amino acid sequence of SEQ ID NO:1, and preferably, said site-directed pegylation is achieved by mutagenesis of the cysteine at position 303 of the arginase; and the average molecular weight of the PEG is about 20K or 30K. Preferably the said cysteine is mutated to alanine, glycine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan or proline; and more preferably, said cysteine is mutated to alanine,

In one aspect of the present invention, the pegylated arginase is site-directed pegylated at position 168 of amino acid sequence of SEQ ID NO:1; and preferably, said site-directed pegylation is achieved by mutagenesis of the cysteines at positions 45 and 303 of the arginase, and the average molecular weight of the PEG is about 40K. Preferably the said cysteines are mutated to alanine, glycine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan or proline; and more preferably, the said cysteines are mutated to alanine,

In one aspect of the present invention, the pegylated arginase is site-directed pegylated at position 45 of amino acid sequence of SEQ ID NO:1; and preferably, said site-directed pegylation is achieved by mutagenesis of the cysteine at positions 168 and 303 of the arginase, and the average molecular weight of the PEG is about 40K. Preferably the said cysteines are mutated to alanine, glycine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan or proline; and more preferably, said cysteines are mutated to alanine,

The present invention provides a pegylated arginase having a purity of more than 70%; preferably, the purity is more than 80%; and more preferably, the purity is more than 90%. In the present invention, the term “purity of pegylated arginase” means the percentage of the pegylated arginase (i.e., arginase covalently linked with PEG) in the total arginase (arginase covalently linked with PEG, arginase not covalently linked with PEG and polymeric pegylated arginase). Generally, the product needs further purification because it contains arginase not covalently linked with PEG and polymeric pegylated arginase. Commonly used means of purification technique are known in the art, including the cation exchange chromatography, etc.

The pegylated arginase of the present invention has a half-life of at least 0.5 day; preferably, said arginase has a half-life of at least 2.5 days; and more preferably, said arginase has a half-life of at least 3.5 days in the blood or serum. The half-life of the arginase can be measured with the known and commonly used method in the art. There is certain relationship between the data measured for the half-life of arginase in the blood or serum and the animal model used. The half-life data obtained in an animal with higher metabolic rate (e.g., rat) is generally shorter than that of with lower metabolic rate (e.g., human).

The present invention provides a method for preparing a pegylated arginase, wherein the pegylation is site-specific. In the pegylated arginase of the present invention, the PEG molecule is conjugated with the specific group on the arginase to achieve the site-specific pegylation. In the pegylated arginase of the present invention, each arginase molecule is linked with at least one PEG molecule to form the pegylated arginase.

In the present invention, site-directed pegylation is achieved by chemical modification, wherein said modification is carried out by covalently conjugating PEG molecules with the groups on the arginase in the presence of coupling reagent. Said chemical modification can be specific, that is, the PEG molecule only conjugates with specific amino acid residue of the arginase using reagents that bind with specific group.

The present invention also provides the application/use of the arginase or the pegylated arginase mentioned above or the pharmaceutical composition containing the same in treating an arginase-related disease. Said arginase related diseases known in the field include conditions/disease/disorders related with arginine level in the body of mammals. Such conditions/disease/disorders include hyperargininemia. Due to arginase deficiency, the arginine in the body of a subject can not be degraded into urea and participate in the ornithine metabolism cycle, so that the blood arginine level can be 7 to 10 times higher than normal blood value, at the same time the arginine level in the cerebrospinal fluid and urine and urea excretion of creatinine are also increased. Moreover, said conditions/disease/disorders include arginine-dependent hyperplasia or tumor. By studying the effect on cell growth, it was found that upon removal of arginine, the normal cells in the cell cycle G0 phase would enter a resting state and remain viable for several weeks without significant damage; when the concentration of arginine returned to normal level, the cells would return to normal cell cycle. Cells in proliferation or tumors, however, would proceed past the ‘R’ point of the cell cycle G1 phase to enter S phase at the deprivation of arginine, and undergo apoptosis soon. The apoptosis of hyperplasia cells or tumor cells as a result of arginine deficiency is irreversible. Therefore, scientists began to consider treating hyperplasia or tumor by controlling the level of arginine in the body.

The present invention also provides a pharmaceutical composition, wherein the active ingredient of the said pharmaceutical composition of the present invention is the arginase or the pegylated arginase as described above. The formulation of said pharmaceutical composition can be in the form of solid, solutions, emulsions, dispersions, micelles, liposomes, wherein the formulation contains one or more of the human arginase or pegylated arginase of the present invention as active ingredient and admixed with organic or inorganic carrier or excipient suitable for parenteral applications. Furthermore, adjuvants, stabilizers, thickeners, coloring agents and perfumes can be included. One or more isolated and substantially pure human arginases or pegylated arginases of the present invention are comprised as active ingredient in a sufficient amount to produce the desired effect on the conditions or diseases. The pharmaceutical compositions may be formulated in forms suitable for oral administration, such as tablets, pills, lozenges, hydration or oil based suspensions, dispersed powders or granules, emulsions, hard or soft capsules, or syrups. Formulations for oral administration may be encapsulated in accordance with techniques known in the art to slow down the decomposition and absorption in the gastrointestinal tract, thereby providing sustained effects for a longer time. The formulation may also be a sterile injectable solution or suspension form. The said suspension can be prepared according to methods known in the art by using dispersing or wetting agents and suspending agents.

The pharmaceutical composition of the present invention can be further formulated into a solid, liquid, suspension, micelle or liposome form. In one aspect of the present invention, the pharmaceutical composition is formulated into an oral or injectable form.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of wide type arginase I.

FIG. 2 shows SDS-polyacrylamide gel electrophoresis analysis of human arginase I under reducing condition.

FIG. 3 shows the expression of human arginase by High Pressure Liquid Chromatography analysis.

FIG. 4 shows the test results of wild type and mutant arginase I with reducing and non-reducing SDS-polyacrylamide gel electrophresis.

FIG. 5 shows SDS-polyacrylamide gel electrophoresis analysis of purified mutant arginase I (rhArgI-A303, rhArgI-A168/303 and rhArgI-A45/303) under reducing and non-reducing conditions.

FIG. 6 shows SDS-polyacrylamide gel electrophoresis analysis of purified mutant arginase I (rhArgI-A45/168 and rhArgI-A45/168/303) under reducing and non-reducing conditions.

FIG. 6 a shows non-reducing SDS-polyacrylamide gel electrophoresis analysis of purified mutant arginase I (rhArgI-A45/168 and rhArgI-A45/168/303).

FIG. 6 b shows reducing SDS-polyacrylamide gel electrophoresis analysis of purified mutant arginase I (rhArgI-A45/168 and rhArgI-A45/168/303).

FIG. 7 shows non-reducing SDS-polyacrylamide gel electrophoresis analysis of pegylated human arginase I.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

This invention will be further illustrated using the embodiments described below. It should be understood that although these embodiments illustrate the preferred embodiments of the present invention, they are only given by ways of illustrated samples. With reference to the above description and embodiments, the skilled in the art can determine the essential characteristics of the invention, change and modify the invention in various ways in adaptation to other uses and conditions without deviating from the spirit and scope of the invention. Therefore, other than those shown and described herein, according to the foregoing, any changes/modifications made to this invention will be apparent to the skilled in the art and these modifications are also intended to form part of the scope of the appended claims.

EXAMPLE 1 Expression and Construction of His-tag-Free Recombinant Human Arginase I Plasmid pET30a (+)-rharginase-V

Human arginase I gene sequence was published in 1987 (Haraguchi. Y. et al., 1987, Proc. Natl. Acad. Sci, 84, 412-415). Human arginase gene was amplified by Polymerase Chain Reaction (PCR) using human liver 5′ stretch plus cDNA library (clontech) as template and primers ARG-V (+) and ARG-V (−) designed according to the aforesaid arginase I sequence. Primer ARG-V (+) contains an NdeI restriction endonuclease site, and and ARG-V (−) primer contains a XhoI restriction endonuclease site. Using common laboratory techniques of molecular biology, human arginase I PCR product was obtained and confirmed by argarose gel electrophoresis. The above amplified human arginase I PCR product and commercially available expression vector pET30a (+) were digested with restriction endonucleases, NdeI and XhoI (Promega) separately at 37° C. for 1.5 h. The digested fragments were ligated overnight at 16° C. by T4 DNA ligase, and the resulting ligated recombinant plasmid was transformed into DH5α E. coli competent cells. The transformed cells were selected by plating and culturing on a kanamycin (30 μg/ml) containing LB agar plate. Plasmids containing the correct insert were identified by restriction endonuclease digest assay. The correct recombinant human arginase I expression plasmid, hereafter known as pET30a (+)-rharginase-V, was analyzed by sequencing to ensure the correct sequence and insertion of human arginase I gene. The insert size is 969 base pair in length. As shown in FIG. 1, it has a nucleotide sequence of SEQ ID No. 2.

ARG-V(+): 5′ GGAATTCCATATGAGCGCCAAGTCCAGAACCATAG 3′           NdeI ARG-V(−): 5′ CCGCTCGAGTTATTACTTAGGTGGGTTAAGGTAGTCAATAGG 3′         XhoI

EXAMPLE 2 Expression of His-tag-Free Recombinant Human Arginase I Plasmid DNA pET30a (+)-rharginase-V and Purification of Target Protein

One-hundred μl of competent BL21(DE3) cells were thawed on ice. One μl of pET30a (+)-V recombinant plasmid was added to the competent cells and incubated on ice for 30 min. The mixture was then heat shock treated in a 42° C. water bath for 90s followed by a further incubation on ice for 2 min. Five-hundred μl of LB broth was added to the transformed cells and shake-cultured at 37° C., 150 rpm for 1 h. After incubation, 2000 of the transformed cell suspension was plated and spread on a kanamycin LB agar plate, which is then incubated upside-down in a 37° C. incubator for 16 h.

Single colony transformed with recombinant plasmid was selected, transferred into 25 ml of LB broth and shake-cultured at 37° C., 150 rpm until the optical density 600 nm (OD600 nm) reaching 0.6-0.8. A final concentration of 0.2 mM IPTG was added to the culture to induce target protein expression for 3 h. Target protein expression of the selected clones was analyzed using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The clones with the higher protein expression were stored in glycerol stock as engineered bacteria.

The engineered bacteria cells were fed-batch cultured in a 15 L fermentor. When OD600 nm reaches 12-13, a final concentration of 0.2 mM IPTG was added into the culture to induce target protein expression for 3-4 h. Induced bacteria cells were collected and lysed. The cell lysate was centrifuged and the supernatant was collected for subsequent target protein separation and purification.

Target protein purification was performed using CM cation liquid chromatography. Tris-HCl was used to equibrate the column prior to sample loading. The lysate supernatant of bacterial cell was loaded into the column, followed by washing with three column volumes of equilibration buffer to remove weakly associated or non-specifically bound impurities. When the reading of UV280 nm stabilized, the target protein was eluted using an elution buffer containing Tris-HCl and NaCl. The protein peaks eluted from the column were collected and analyzed by SDS-PAGE and High Pressure Liquid Chromatography (HPLC).

Experimental results were illustrated in FIGS. 2 and 3.

FIG. 2 shows the result of SDS-PAGE analysis of human arginase I expression level. The samples in each lane are as follow:

1. protein molecular weight standard; 2. bacterial lysate supernatant; 3. purified His-tag-free human arginase I; 4. CM cation exchange column flow through.

FIG. 3 shows the result of HPLC analysis of purified human arginase I.

As shown in FIGS. 2 and 3, the purity of arginase obtained via the above chromatography procedure is more than 90%.

EXAMPLES 3 Site-Directed Mutagenesis of Human Arginase I (rhArgI)

Site-directed mutations were introduced at positions 45, 168 and 303 of amino acid sequence of human arginase I using QuickChange site-directed mutagenesis kit (Stratagene). Recombinant plasmid pET30a-rharginase I-V containing wild type human arginase was used as template. Codons that encode for cysteine (TGT) at positions 45, 168 and 303 were independently mutated to codons that encode for alanine (GCT), resulting in single, double or triple-site mutations. The primers intended to introduce the replacement codon GCT at positions 45, 168, and 303 of human arginase I are shown as below:

ARG-m45(+): 5′ GAGAAACTTAAAGAACAAGAGGCTGATGTGAAGGATTATGGGG 3′ ARG-m45(−): 5′ CCCCATAATCCTTCACATCAGCCTCTTGTTCTTTAAGTTTCTC 3′ ARG-m168(+): 5′ GATTCTCCTGGGTGACTCCCGCTATATCTGCCAAGGATATTG 3′ ARG-m168(−): 5′ CAATATCCTTGGCAGATATAGCGGGAGTCACCCAGGAGAATC 3′ ARG-m303(+): 5′ GTTGCAATAACCTTGGCTGCTTTCGGACTTGCTCGGG 3′ ARG-m303(−): 5′ CCCGAGCAAGTCCGAAAGCAGCCAAGGTTATTGCAAC 3′

PCR was conducted in accordance with the instruction provided in the mutagenesis kit. Non-mutated parental DNA template was digested with restriction endonuclease DpnI. The mutated plasmids were then transformed into competent cells and confirmed by sequencing. Clones that were transformed with the mutant arginase plasmid containing the mutation of cysteine encoding codon (TGT) to alanine encoding codon (GCT) were selected and expanded in LB broth culture. Target mutant plasmids were isolated using Wizard Plus mini prep kit.

Mutant human arginase I with single cysteine to alanine mutation at amino acid position 303, 168 or 45 was represented as rhArgI-A303, rhArgI-A168 or rhArgI-A45 respectively. Mutant human arginase I with double cysteine to alanine mutations at amino acid positions 168 and 303, at amino acid positions 45 and 303 and at amino acid positions 45 and 168 were represented as rhArgI-A168/303, rhArgI-A45/303 and rhArgI-A45/168 respectively. Mutant human arginase I with three cysteine to alanine mutations at amino acid positions 45, 168 and 303 was represented as rhArgI-A45/168/303. Plasmids containing the above arginase mutant inserts were represented as pET30a(+)-rhArgI-A303, pET30a(+)-rhArgI-A168, pET30a(+)-rhArgI-A45, pET30a(+)-rhArgI-A168/303, pET30a(+)-rhArgI-A45/303, pET30a(+)-rhArgI-A45/168 or pET30a(+)-rhArgI-A45/168/303.

One-hundred μl of competent BL21(DE3) cells were thawed on ice. One μ of the mutant construct was added to the BL21(DE3) competent cells and incubated on ice for 30 min. The mixture was then heat shock treated at 42° C. for 90 s, followed by a further incubation on ice for 2 min. Five-hundred μl of LB broth was added to the transformed cells and shake-cultured at 37° C., 150 rpm for 1 h. After incubation, 2000 of the transformed cell suspension was plated and spread on a kanamycin LB agar plate and incubated upside-down at 37° C. for 16 h.

Single colony transformed with recombinant plasmid was selected, transferred into 25 ml of LB broth, and shake-cultured at 37° C., 150 rpm, until the optical density at 600 nm (OD600nm) reaching 0.6-0.8. A final concentration of 0.2mM IPTG was added to the culture to induce target protein expression for 3 h. Target protein expression in the selected clones was analyzed using SDS-PAGE. The clones with higher protein expression were stored in glycerol stock as engineered bacteria.

EXAMPLE 4 Expression and Protein Purification of Site Specific Mutated Arginase I

Engineered E. coli cells transformed with mutant arginase I (rhArgI) plasmids (pET30a(+)-rhArgI-A303, pET30a(+)-rhArgI-A168, pET30a(+)-rhArgI-A45, pET30a(+)-rhArgI-A168/303, pET30a(+)-rhArgI-A45/303, pET30a(+)-rhArgI-A45/168 or pET30a(+)-rhArgI-A45/168/303) were fed-batch cultured in a 15L fermentor. When OD600 nm reached 12-13, a final concentration of 0.2 mM IPTG was added to the culture to induce target protein expression for 3-4 h. The bacterial cells were collected and lysed. The cell lysate was centrifuged and the supernatant was collected for subsequent protein isolation and purification.

Target protein purification was performed using CM cation exchange column. Tris-HCl was used to equilibrate the column prior to sample loading. The lysate supernatant of bacterial cells was loaded into the column, followed by washing with three times column volume of equilibration buffer to remove weakly associated or non-specifically bound impurities. When the reading of UV280 nm stabilized, target protein was eluted using an elution buffer containing Tris-HCl and NaCl. The protein peaks eluted from the column were collected to obtain mutant arginase I rhArgI-A303, rhArgI-A168, rhArgI-A45, rhArgI-A168/303, rhArgI-A45/303, rhArgI-A45/168 and rhArgI-A45/168/303.

Purity of the collected protein samples was analyzed by SDS-PAGE and HPLC. Using the above chromatography procedure, the purity of arginase was determined to be more than 90%.

EXAMPLE 5 Activity of Site-Specific Mutant Arginase I (rhArgI)

The activity of arginase was determined by a spectrophotometric assay coupled with urease and glutamate dehydrogenase, which is shown by schematic diagram below. NADPH has optimal absorbance at the wavelength of 340 nm. When NADPH is oxidised to NADP+, the absorbance at 340 nm decrease. The activity of arginase can be correlated and determined by monitoring the decrease in absorbance at 340 nm together with the molar extinction coefficient of NADPH (ΔE340=6220 M⁻¹ cm⁻¹).

One Unit (U) of arginase activity was defined as the release of 1 μmol urea under the condition of 30° C., pH 8.3.

Specific activity of arginase was calculated using the following equation:

Specific activity(U/mg)=[(ΔA/Δt)×(1/ε)×10⁶×(1/2)]/[E]

ΔA=Differences in absorbance at 340 nm

ε=NADPH Michaelis-Menten constant(Km) (6220 M⁻¹cm⁻¹)

[E]=enzyme concentration in the reaction mixture (mg/mL)

Using the above assay, the specific activity of various mutant arginase I (rhArgI-A303, rhArgI-A168/303, rhArgI-A45/303, rhArgI-A45/168 and rhArgI-A45/168/303) were determined to be 747±63 U/mg, 814±91 U/mg, 786±58 U/mg, 782±19 U/mg, and 759±68 U/mg respectively. The specific activity of wild type human arginase I was determined to be 491±42 U/mg, demonstrating a significant increase of arginase activity after the non-polar nonionic amino acid cysteines at positions 168/303, 45/303, 45/168 and 45/168/303 were mutated to non-polar amino acid alanines.

EXAMPLE 6 SDS-PAGE Analysis of Wild Type and Mutant Arginase I (rhArgI)

The polyacrylamide gel was prepared using traditional method. Samples were treated under reducing (i.e. β-mecaptoethanol was included in the sample loading buffer to destroy inter and intra molecular disulfide bonds) or non-reducing condition prior to analysis to decipher the intramolecular disulfide bridge formation of wild type and various mutant arginase I using 12% polyacrylamide gel electrophoresis (FIG. 4, 5, 6A, 6B).

FIG. 4 shows the result of SDS-PAGE analysis of wild type arginase I after treatment under reducing and non-reducing conditions. The samples in the lanes are as follow: Lane 1 and 3: bacterial cell lysate supernatant; Lane 2: purified His-tag-free arginase (non-reducing); M: protein molecular weight standard; Lane 4: purified His-tag-free human arginase I (reducing).

FIG. 5 shows the result of SDS-PAGE analysis of purified mutant arginase I (rhArgI-A303, rhArgI-A168/303 and rhArgI-A45/303) under reducing and non-reducing conditions. The samples in each lane are as follow: Lane 1: mutant arginase I, rhArgI-A303 (non-reducing); Lane 2: mutant arginase I, rhArgI-A168/303 (non-reducing); Lane 3: mutant arginase I, rhArgI-A45/303 (non-reducing); M: protein molecular weight standard; Lane 4: mutant arginase I, rhArgI-A303 (reducing); Lane 5: mutant arginase I, rhArgI-A168/303 (reducing); Lane 6: mutant arginase I, rhArgI-A45/303 (reducing).

FIG. 6 a shows the result of SDS-PAGE analysis of purified mutant arginase I (rhArgI-A45/168 and rhArgI-A45/168/303) under non-reducing condition. The samples in each lane are as follow: M: protein molecular weight standard; Lane 1: mutant arginase I, rhArgI-A45/168/303; Lane 2: mutant arginase I, rhArgI-A45/168.

FIG. 6 b shows the result of SDS-PAGE analysis of purified mutant arginase I (rhArgI-A45/168 and rhArgI-A45/168/303) under reducing condition. The samples in each lane are as follow: M: protein molecular weight standard; Lane 1: mutant arginase I, rhArgI-A45/168/303; Lane 2: mutant arginase I, rhArgI-A45/168.

As revealed from the SDS-PAGE analysis of arginase I under reducing and non-reducing conditions, wild type human arginase I have other conformations under the stated experimental conditions of this invention. Under such condition, when cysteine at position 303 was mutated to alanine, other conformations were also present. However, when either two (rhArgI-A168/303, rhArgI-A45/303, or rhArgI-A45/168) or all of the three cysteine residues at amino acid positions 45, 168 and 303 were mutated to alanine, only one conformation was detected under non-reducing condition (FIG. 5 and FIG. 6 A). Not limited by any known theories, the inventor believes the presence of multiple conformations for wild type human arginase I at non-reducing SDS-PAGE is probably due to intramolecular disulfide bond formation that is contributed by cysteines 45, 168 and 303 under certain conditions. When only cysteine 303 was mutated to alanine, such conformation of arginase I mutant could also be detected by SDS-PAGE under non-reducing condition possibly due to the formation of intramolecular disulfide bonds between unmutated cysteines 45 and 168. When either two or all of the three cysteine residues at positions 45, 168 and 303 were mutated (rhArgI-A168/303, rhArgI-A45/303, rhArgI-A45/168 or rhArgI-A45/168/303) all possibilities of disulfide bond formation will be abolished, resulting in only one conformation being detected in the SDS-PAGE analysis.

EXAMPLE 7 Site-Directed Pegylation of Mutated Arginase I (rhArgI) and Purification of Pegylated Protein

Site-specific arginase I mutants (rhArgI-A303, rhArgI-A168/303 or rhArgI-A45/303) were mixed separately with methoxy polyethylene glycol maleimide in a molar ratio range of 1:5-1:10 in 20 mM PBS buffer (pH 7.0), wherein said methoxy polyethylene glycol maleimide has a molecular weight of 20K, 30K or 40K respectively. The 40K methoxy polyethylene glycol maleimide was Y-shape branched, consisting of two polyethylene glycol chains. The pegylation reaction was carried out at room temperature for 2-4 h. At the end of the reaction, the end products were stored in a 4° C. refrigerator.

Site-specific pegylated human arginase I was isolated and purified using Macro SP matrix cation exchange column to remove residual unreacted proteins and PEGs. Phosphate buffer and NaCl (1M) containing phosphate buffer were used as equilibration and elution buffer respectively. Prior to sample loading, pegylated protein samples were diluted with distilled water until the sample electric conductivity is identical to that of the equilibration buffer; and column was equilibrated with five times column volume of equilibration buffer. After sample loading, the column was further washed with five times column volume of equilibration buffer and eluted with 35% elution buffer. The eluant containing protein peaks were collected and desalted using G25 column. Target proteins were collected and analyzed by SDS-PAGE as shown in FIG. 7.

A303-M20K(2): Site-directed mutated arginase I (rhArgI-A303) was reacted with methoxy polyethylene glycol maleimide-20 kDa (mPEG-MAL-20K). The site-directed pegylated human arginase I was purified by Macrocap SP matrix cation exchange column. The percentage of the pegylated human arginase I conjugated with two 20 kDa mPEG-MAL molecules (referred to A303-M20K(2)) at cysteines 45 and 168 accounts for more than 70% of the total reaction product.

A303-M30K(2): Site-directed mutated arginase I (rhArgI-A303) was reacted with methoxy polyethylene glycol maleimide-30 kDa (mPEG-MAL-30K). The site-directed pegylated human arginase I was purified by Macrocap SP matrix cation exchange column. The percentage of the pegylated human arginase I conjugated with two 30 kDa mPEG-MAL molecules (referred to A303-M30K(2)) at cysteines 45 and 168 accounts for more than 70% of the total reaction product.

A168/303-Y40K: Site-specific mutated arginase I (rhArgI-A168/303) was reacted with Y-shape branched polyethylene glycol maleimide 40K (Y-MAL-40K). Pegylated human arginase I was purified by Macrocap SP matrix cation exchange column. The pegylated arginase I protein conjugated with one PEG chain Y-MAL-40K at cysteine 45 (A168/303-Y40K) accounts more than 85% of the total reaction products.

A45/303-Y40K: Site-specific mutated arginase I (rhArgI-A45/303) was reacted with Y-shape branched polyethylene glycol maleimide 40K (Y-MAL-40K). Pegylated human arginase I was purified by Macrocap SP matrix cation exchange column. The pegylated arginase I protein conjugated with one PEG chain Y-MAL-40K at cysteine 168 (A45/303-Y40K) accounts for more than 85% of the total reaction products.

Using the coupled spectrophotometric assay previously described, the activity of different pegylated human arginase I was measured and determined by changes in absorbance of NADPH. Test results showed that the pegylated products preserved the arginase activity, wherein the specific activities of A303-M20K(2), A303-M30K(2), A168/303-Y40K and A45/303-Y40K were all higher than 400 U/mg, ranging 400-800 U/mg.

EXAMPLE 7 In-Vivo Pharmacokinetic Analysis of Site-Directed Pegylated Human Arginase I

Pharmacokinetics profiles of different pegylated human arginase I were evaluated after single intravenous administration to Sprague Dawley rats at a dosage of 3 mg/kg. These studies were conducted by Pharmalegacy Laboratories Limited (Shanghai). Blood samples were collected pre-dose, 2 min, 1 h, 4 h, 24 h, 72 h, 120 h, 168 h and 240 h after dosing for serum arginase and arginine analysis. Approximately 0.4 mL of blood samples were collected at each time-point from orbital vein after anesthesia by isoflurane and transferred to a 2-mL centrifuge tube. Samples were placed at room temperature for 30 min until centrifugation at 4,000 g for 15 min at 4° C.

Serum concentrations of arginase I were determined by a quantitative sandwich enzyme immunoassay with kits provided by Shanghai ExCell Biology, Inc. Anti-human arginase I monoclonal antibody was coated on the surface of the wells of the ELISA plate. Samples or protein standards of human arginase I were added separately into the wells of the ELISA plate (1000 well), which was then covered with sealer tape and incubated at 37° C. for 90 min to allow immunocomplex formation between monoclonal antibody and human arginase I. After incubation, the plate was washed five times and rabbit anti-human arginase I polyclonal antibodies (1000 well) was added to the wells. It was covered with sealer tape again and further incubated at 37° C. for 60 min. After the second incubation, the plate was washed again for five times, HRP-conjugated goat anti-rabbit IgG (1000 well) was added to each well and co-incubated for 30 min. After another five times washing, chromogenic substrate was added into the wells and incubated in the dark for 10-15 min. Stop solution was added to each well and mixed well. The optical density at 450 nm was measured within 10 min after adding the stop solution. The standard curve was generated using quadratic fit method for regression equation and the arginase I concentration of each samples was calculated using the measured OD value. The PK parameters were derived using WinnoLin with a non-compartmental assay method. The half-life (T_(1/2)) values of various forms of site-specific pegylated human arginase I, A303-M20K(2), A303-M30K(2), A168/303-Y40K and A45/303-Y40K were determined to be 28.0±4.5, 28.4±8.4, 27.2±3.8, and 15.1±0.6 h respectively.

TABLE 1 The pharmacokinetic parameters (average ± standard deviation; n = 6) of single dose administration of pegylated human arginase I in rat. T_(1/2) C_(max) AUC_(last) AUC_(INF) (h) (μg/mL) (h*μg/mL) (h*μg/mL) A303- 28.0 ± 4.5 41.5 ± 9.5 1603.5 ± 187.5 1614.5 ± 188.6 M20K(2) A303- 28.4 ± 8.4 32.4 ± 3.1 1113.6 ± 116.8 1192.9 ± 121.2 M30K(2) A168/303- 27.2 ± 3.8 45.2 ± 5.1 2077.5 ± 160.6 2083.9 ± 163.3 Y40K A45/303- 15.1 ± 0.6 105.2 ± 66.9 548.1 ± 50.3 564.5 ± 50.8 Y40K

This invention uses site-directed mutagenesis to modify either one, or two or all of the three cysteine residues at positions 45, 168 and 303 of human arginase I, wherein said modification is conducted by replacement of the codon encoding for cysteine to other amino acids, particularly, alanine, to obtain human arginase with higher specific activity. In addition, the present invention provides a novel site-directed pegylated human arginase, which does not contain any potentially immunogenic his-tag protein sequence. Said pegylated human arginase was pegylated by conjugating 20, 30 or 40 kDa polyethylene glycol maleimide respectively to cysteine residue 45, 168 or 303. The pegylated human arginase produced in this way has a reduced glomerular filtration rate. Furthermore, the pegylation site of the arginase in conjugation with PEG was definite and the pegylation product is more homogenous and easier for purification, favorable for quality control of mass production. The pegylated arginase provided in the present invention has a significantly increased half-life in mammals, which can be as long as 15-28 h in sera of rats.

Unless otherwise stated, the practice of this invention involves the use of common techniques that are employed in biotechnology, organic chemistry, or inorganic chemistry, etc. Apparently, other than the description and embodiments mentioned above, there are other means can be used to implement the present invention. Any changes or modifications under the scope of this invention will be apparent to the skilled in the art. According to the teachings of the present invention, many changes and modifications are possible and are hence classified under the coverage of this invention. The patents mentioned in this article, their applications and scientific papers are all incorporated into this article. 

What is claimed is:
 1. (canceled)
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled)
 6. A pegylated arginase, wherein said arginase is human arginase I; the pegylation of said arginase is site-specific and any one or two of the cysteines at positions 45, 168 and 303 of amino acid sequence of SEQ ID NO:1 is/are pegylated; said site-specific pegylation is achieved by site-directed mutation of cysteine to other amino acid at any one or two of said cysteine positions which is/are not to be pegylated, and followed by site-specific pegylation of mutant arginase at the cysteine residue; wherein in said arginase, (1) any one or two of the cysteines at positions 45, 168 and 303 of said SEQ ID NO:1 is/are mutated to alanine; or (2) substitution, deletion, insertion, addition or inversion of one or more amino acids is introduced in said SEQ ID NO:1; wherein said arginase has activity.
 7. (canceled)
 8. The pegylated arginase of claim 6, wherein any one of said cysteines at positions 45, 168 and 303 of said SEQ ID NO:1 is mutated to alanine.
 9. The pegylated arginase of claim 6, wherein said arginase is conjugated with polyethylene glycol (PEG): said PEG has an average molecular weight of 20K, 30K or 40K.
 10. The pegylated arginase of claim 6, wherein said arginase has a half-life of at least 0.5 day.
 11. (canceled)
 12. (canceled)
 13. A pharmaceutical composition for treating an arginase-related disease comprising said pegylated arginase of claim
 6. 14. A method for treating an arginase-related disease, comprising administrating said pegylated arginase of claim
 6. 15. The pegylated arginase of claim 6, wherein said cysteine at position 303 of said SEQ ID NO:1 is mutated to alanine.
 16. The pegylated arginase of claim 15, wherein the site-specific pegylation is carried out at Cys45 and Cys168; said PEG has an average molecular weight of 20K or 30K.
 17. The pegylated arginase of claim 6, wherein said cysteines at positions 45 and 303 of said SEQ ID NO:1 are mutated to alanine.
 18. The pegylated arginase of claim 17, wherein the site-specific pegylation is carried out at Cys168; said PEG has an average molecular weight of 40K.
 19. The pegylated arginase of claim 6, wherein said cysteines at positions 168 and 303 of said SEQ ID NO:1 are mutated to alanine.
 20. The pegylated arginase of claim 19, wherein the site-specific pegylation is carried out at Cys45; said PEG has an average molecular weight of 40K.
 21. The pegylated arginase of claim 6, wherein said site-specific pegylation is cysteine-specific and carried out at the cysteine residue; said cysteine-specific pegylation is achieved by covalently linking polyethylene glycol with the thiol group of cysteines on said arginase by methoxy polyethylene glycol maleimide.
 22. The pegylated arginase of claim 6, wherein the purity of said pegylated arginase is above 70%.
 23. The pharmaceutical composition of claim 13, wherein said disease is selected from hyperargininemia and arginine-dependent hyperplasia or tumor.
 24. The method of claim 14 wherein said disease is selected from hyperargininemia and arginine-dependent hyperplasia or tumor. 